Pursuant to 37 C.F.R. 1.71(e), Applicants note that a portion of this disclosure contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.
The present invention is in the field of transmembrane potential measurement using Nernstian dyes, e.g., in microfluidic systems.
Cell-based assays are often preferred for an initial screening of biologically active compounds, due to the approximation of in vivo systems by cells, combined with their capability to be rapidly screened. A variety of cell responses to stimuli can be detected, including cell death, transporter function and response to chemical stimuli.
The distribution of a permeable ion between the inside and outside of a cell or vesicle depends on the transmembrane potential of the cell membrane. In particular, for ions separated by a semi permeable membrane, the electrochemical potential difference (xcex94xcexcj) which exists across the membrane, is given by xcex94xcexcj=2.3 RT log [jI]/[jo]+zERF, where R is the universal gas constant, T is an absolute temperature of the composition, F is Faraday""s constant in coulombs, [jI] is the concentration of an ion (j) on an internal or intracellular side of the at least one membrane, [jo] is the concentration of j on an external or extracellular side of the at least one membrane, z is a valence of j and ER is a measured transmembrane potential. Thus, the calculated equilibrium potential difference (Ej) for ion j=xe2x88x922.3RT(zF)xe2x88x921log[jI]/[jo] (this is often referred to as the Nernst equation). See, Selkurt, ed. (1984) Physiology 5th Edition, Chapters 1 and 2, Little, Brown, Boston, Mass. (ISBN 0-316-78038-3); Stryer (1995) Biochemistry 4th edition Chapters 11 and 12, W.H. Freeman and Company, N.Y. (ISBN 0-7167-2009-4); Haugland (1996) Handbook of Fluorescent Probes and Research Chemicals Sixth Edition by Molecular Probes, Inc. (Eugene Oreg.) Chapter 25 (Molecular Probes, 1996) and http://www.probes.com/handbook/sections/2300.html (Chapter 23 of the on-line 1999 version of the Handbook of Fluorescent Probes and Research Chemicals Sixth Edition by Molecular Probes, Inc.) (Molecular Probes, 1999) and Hille (1992) Ionic Channels of Excitable Membranes, second edition, Sinauer Associates Inc. Sunderland, Mass. (ISBN 0-87893-323-9) (Hille), for an introduction to transmembrane potential and the application of the Nernst equation to transmembrane potential. In addition to the Nernst equation, various calculations which factor in the membrane permeability of an ion, as well as Ohm""s law, can be used to further refine the model of transmembrane potential difference, such as the xe2x80x9cGoldmanxe2x80x9d or xe2x80x9cconstant fieldxe2x80x9d equation and Gibbs-Donnan equilibrium. See Selkurt, ed. (1984) Physiology 5th Edition, Chapter 1, Little, Brown, Boston, Mass. (ISBN 0-316-78038-3) and Hille at e.g., chapters 10-13.
Increases and decreases in resting transmembrane potentialxe2x80x94referred to as membrane depolarization and hyperpolarization, respectivelyxe2x80x94play a central role in many physiological processes, including nerve-impulse propagation, muscle contraction, cell signaling and ion-channel gating. Potentiometric optical probes (typically potentiometric dyes) provide a tool for measuring transmembrane potential and changes in transmembrane potential over time (e.g., transmembrane potential responses following the addition of a composition which affects transmembrane potential) in membrane containing structures such as organelles (including mitochondria and chloroplasts), cells and in vitro membrane preparations. In conjunction with probe imaging techniques (e.g., visualization of the relevant dyes), these probes are employed to map variations in transmembrane potential across excitable cells and perfused organs.
For example, the plasma membrane of a cell at rest typically has a transmembrane potential of approximately xe2x88x9220 to xe2x88x9270 mV (negative inside) as a consequence of K+, NA+ and Clxe2x88x92 concentration gradients (and, to a lesser extent, H+, Ca2+, and HCO3xe2x88x92) that are maintained by active transport processes. Potentiometric probes are important tools for studying these processes, as well as for visualizing, e.g., mitochondria (which exhibit a large transmembrane potential of approximately xe2x88x92150 mV, negative inside matrix), and for cell viability assessment. See, Molecular Probes (1996) chapter 25 and the references cited therein.
Potentiometric probes include cationic or zwitterionic styryl dyes, cationic rhodamines, anionic oxonols, hybrid oxonols and merocyanine 540. The class of dye determines factors such as accumulation in cells, response mechanism and cell toxicity. See, Molecular Probes 1999 and the reference cited therein; Plasek et al. (1996) xe2x80x9cIndicators of Transmembrane potential: a Survey of Different Approaches to Probe Response Analysis.xe2x80x9d J Photochem Photobiol: Loew (1994) xe2x80x9cCharacterization of Potentiometric Membrane Dyes.xe2x80x9d Adv Chem Ser 235, 151 (1994); Wu and Cohen (1993) xe2x80x9cFast Multisite Optical Measurement of Transmembrane potentialxe2x80x9d Fluorescent and Luminescent Probes for Biological Activity, Mason, Ed., pp. 389-404; Loew (1993) xe2x80x9cPotentiometric Membrane Dyes.xe2x80x9d Fluorescent and Luminescent Probes for Biological Activity, Mason, Ed., pp. 150-160; Smith (1990) xe2x80x9cPotential-Sensitive Molecular Probes in Membranes of Bioenergetic Relevance.xe2x80x9d Biochim Biophys Acta 1016, 1; Gross and Loew (1989) xe2x80x9cFluorescent Indicators of Transmembrane potential: Microspectrofluorometry and Imaging.xe2x80x9d Meth Cell Biol 30, 193; Freedman and Novak (1989) xe2x80x9cOptical Measurement of Transmembrane potential in Cells, Organelles, and Vesiclesxe2x80x9d Meth Enzymol 172, 102 (1989); Wilson and Chused (1985) xe2x80x9cLymphocyte Transmembrane potential and Ca+2-Sensitive Potassium Channels Described by Oxonol Dye Fluorescence Measurementsxe2x80x9d Journal of Cellular Physiology 125:72-81; Epps et al. (1993) xe2x80x9cCharacterization of the Steady State and Dynamic Fluorescence Properties of the Potential Sensitive dye bis-(1.3-dibutylbarbituric acid) trimethine oxonol (DiBAC4(3) in model systems and cellsxe2x80x9d Chemistry of Physics and Lipids 69:137-150, and Tanner et al. (1993) xe2x80x9cFlow Cytometric Analysis of Altered Mononuclear Cell Transmembrane potential Induced by Cyclosporinxe2x80x9d Cytometry 14:59-69.
Potentiometric dyes are typically divided into at least two categories based on their response mechanism. The first class of dyes, referred to as fast-response dyes (e.g., styrylpyridinium dyes; see, e.g., Molecular Probes (1999) at Section 23.2), operate by a change in the electronic structure of the dye, and consequently the fluorescence properties of the dye, i.e., in response to a change in an electric field which surrounds the dye. Optical response of these dyes is sufficiently fast to detect transient (millisecond) potential changes in excitable cells, e.g., isolated neurons, cardiac cells, and even intact brains. The magnitude of the potential-dependent fluorescence change is often small; fast-response probes typically show a 2-10% fluorescence change per 100 mV.
The second class of dyes, referred to as slow-response or Nemstian dyes (See, e.g., Molecular Probes, 1999 at Section 23.3), exhibit potential-dependent changes in membrane distribution that are accompanied by a fluorescence change. The magnitude of their optical responses is typically larger than that of fast-response probes. Slow-response probes, which include cationic carbocyanines, rhodamines and anionic oxonols, are suitable for detecting changes in a variety of transmembrane potentials of, e.g., nonexcitable cells caused by a variety of biological phenomena, such as respiratory activity, ion channel permeability, drug binding and other factors. The structures of a variety of available slow response dyes are found e.g., at table 25.3 of Molecular Probes (1996).
Many slow, Nernstian dyes such as carbocyanines, rhodamines and oxonols are used to measure transmembrane potential by virtue of voltage-dependent dye redistribution and fluorescence changes resulting from the redistribution. Fluorescence changes which may be caused by redistribution include: a change of the concentration of the fluorophore within the cell or vesicle, a change in the dye fluorescence due to aggregation or a change in dye fluorescence due to binding to intracellular or intravesicular sites. Typically, 10-15 minutes of equilibration time is used to allow the dyes to redistribute across the plasma membrane after changing the transmembrane potential.
Despite the availability of transmembrane potential sensor compositions and assays, there still exists a need for additional classes of dyes and for new assays and techniques for using potentiometric dyes in biological assays. The present invention fulfills these and a variety of other needs which will become apparent upon complete review of the following.
It is surprisingly discovered that membrane permeable cationic nucleic acid staining dyes can be used as potentiometric dyes for measuring changes in transmembrane potential. In addition, it was discovered that using both cationic dyes (including, but not limited to membrane permeable cationic nucleic acid staining dyes) and anionic membrane permeable redistributing dyes for monitoring changes in transmembrane potential increases the dynamic range and sensitivity of transmembrane potential measurements. Compositions comprising these two classes of dyes and a membrane, as well as microfluidic systems for using the dyes to measure transmembrane potential, are provided. Further it was discovered that measuring the time course of dye uptake, rather than equilibrium distributions of the dyes, leads to improvements in signal to noise ratio, speed of the assay and other benefits.
Accordingly, the present invention provides methods of generating optical signals which depend on transmembrane potential or one or more change in transmembrane potential. For example, in one class of embodiments, the methods include providing a first component comprising one or more membrane, adding a cationic membrane permeable nucleic acid staining dye to the first component, and monitoring a first signal output from the cationic membrane permeable nucleic acid staining dye. To monitor changes in transmembrane potential, changes in the first signal output are monitored over time. The first signal output is then correlated with the transmembrane potential to provide an indication of transmembrane potential or changes in transmembrane potential. Typically, the first composition is also contacted with an anionic membrane permeable redistributing dye to increase the sensitivity and dynamic range of the assay.
In one common format, the relevant components are provided in a microfluidic system. For example, a method of producing a signal which is dependent on transmembrane potential is provided, in which a first mixture which includes one or more membranes and one or more voltage sensitive dyes is flowed through a first channel region. At least a first signal output is monitored from at least one of the voltage sensitive dyes, thereby producing a signal which is dependent on the transmembrane potential across the one or more membranes. For example, the voltage sensitive dyes can include one or more membrane permeable redistributing dyes, including one or more ionic dye. The one or more membrane permeable dyes are typically flowed from a source to the first channel region and into contact with the one or more membranes and flow of the membrane permeable labels across the membrane is detected by monitoring the one or more signal outputs from the membrane permeable labels, typically before equilibrium is reached. The mixture can include a cationic dye, a cationic membrane permeable nucleic acid staining dye, an anionic dye and/or a neutral dye. The one or more voltage sensitive dyes can include, e.g., an anionic or cationic dye (or both), including any or all of: Oxonol V, Oxonol VI, DiBAC4(3), DiBAC4(5), DiBAC2(3), a cationic dye, a cationic membrane permeable nucleic acid staining dye, and a SYTO dye such as SYTO 62.
For example, a cationic dye, such as the cationic membrane permeable nucleic acid staining dye, and the first component are flowed through at least a first microfluidic channel comprising flowing the cationic membrane permeable nucleic acid staining dye through the first or second microchannel and into contact with the at least one membrane. An anionic membrane permeable redistributing dye can also be flowed through the first or second channel and into contact with the at least one membrane.
Thus, in microfluidic formats, methods of measuring or monitoring changes in a transmembrane potential are provided. In the methods, a first component which includes one or more membrane is flowed from a source to a first channel region. A labeling composition comprising a membrane permeable label is flowed into contact with the membrane. The membrane is altered in some way that causes an alteration in transmembrane potential, e.g., by changing the ionic composition on one side of the membrane (e.g., inside or outside of a cell) or by changing the permeability of the membrane to ions. The flow of the membrane permeable label across the membrane is monitored by monitoring a first signal output from the membrane permeable label, thereby measuring changes in the transmembrane potential.
The above methods can include contacting the membrane to one or more transmembrane potential modulatory compositions and monitoring an effect of the one or more transmembrane potential modulatory compositions on the transmembrane potential (e.g., by monitoring the first signal), thereby monitoring an effect of the one or more transmembrane potential modulatory compositions on the transmembrane potential. This can be used as a drug screening method for testing potential modulatory compounds for a transmembrane potential modulatory activity. Examples of modulatory compositions include hyperpolarization buffers, depolarization buffers, compounds which alter the ionic permeability of a membrane, and the like. In addition, control modulators (modulators having a known effect on transmembrane potential in the relevant assay) can be compared to test modulators having unknown effects to determine membrane modulatory activity of the test modulators. Dose response curves for either control or test modulators can be determined and the curves compared.
Control and test modulators can affect, e.g., transporter activity, ion channel activity, or other factors which have an effect on transmembrane potential and changes in transmembrane potential. Examples of test and control modulators include a variety of compounds which effect membrane ionic permeability, ionic potential or the like, including neurotoxins (e.g., such as palytoxin), sets of neurotoxins, neurotransmitters, sets of neurotransmitters, proteins, sets of proteins, peptides, sets of peptides, lipids, sets of lipids, carbohydrates, sets of carbohydrates, organic molecules, sets of organic molecules, drugs, sets of drugs, receptor ligands, sets of receptor ligands, antibodies, set of antibodies, cytokines, sets of cytokines, chemokines, sets of chemokines, hormones, sets of hormones, cells, sets of cells and the like.
In general, the time course of dye translocation across the membrane depends on the transmembrane potential across the membrane. Thus, at a selected time (t) after adding a dye to a membrane, the amount of signal from the dye is correlated to transmembrane potentials. Typically, (t) can be less than about 100 seconds. Commonly, (t) is between about 0.1 and 80 seconds, e.g., between about 10 and 70 seconds. A ratio of first and second signals from the cationic and anionic dyes noted above (e.g., over time) can be determined to further refine estimates of changes in transmembrane potential.
Examples of useful dyes include cyclic-substituted unsymmetrical cyanine dyes and other cationic membrane permeable nucleic acid stains. Examples of useful dyes include Blue-fluorescent SYTO dyes, Green-fluorescent SYTO Dyes, Orange-fluorescent SYTO dyes, Red-fluorescent SYTO dyes such as SYTO 62, Pur-1, thiazol, aryl, 2DS-7J1, Hoechst 33258, Hoechst 33342 and hexidium iodide. Common anionic membrane permeable redistributing dyes include anionic bis-isoxazolone oxonol dyes, bis-oxonol dyes and others. For example, the anionic membrane permeable redistributing dye can be e.g., Oxonol V, Oxonol VI, DiBAC4(3), DiBAC4(5) and/or DiBAC2(3). Example dye concentrations in the relevant systems are typically between about 0.01 and about 50 xcexcM. For example, the cationic dye can be SYTO 62, added to the first component to a concentration of between about 0.01 and about 50 xcexcM and the anionic dye can be DiBAC4(3), added to the first or second component at a concentration of between about 0.01 and about 50 xcexcM.
As noted, examples of membranes of interest include cells, organelles, artificial membranes, membrane preparations of artificial or naturally occurring membrane sources, and the like. Membrane preparations can be suspended in any suitable buffer, e.g., a fluid comprising a membrane permeable ion such as NA+, K+, Clxe2x88x92, H+, Ca2+, or HCO3xe2x88x92. In one embodiment, the membrane is present in an intact or live cell such as an animal cell, a plant cell, a fungal cell, a bacterial cell, or the like. For example, the cell can be a mammalian cell such as a primate cell, a rodent cell, a canine cell, a feline cell, or a livestock cell, or can be e.g., an insect cell or other animal cell. The cell can be a cultured cell such as a THP-1 cell, a COS cell, a CHO cell, a HEK cell, a jurkat cell, a xcex2RL cell, a HeLA cell, an NIH 3T3 cell, an RBL-2H3 cell, or the like. The cell can also be a primary cell such as a cell isolated from endoderm, ectoderm, mesoderm, differentiated tissue, undifferentiated tissue, partially differentiated tissue, blood, peripheral blood, nerve, muscle; skin, bone, or the like.
Typically, signal outputs from dyes are detected by monitoring one or more fluorescent emission produced by the relevant dye. This can be performed spectrophotometrically, optically or, e.g., via microscopy.
In one aspect, the invention provides a microfluidic device for monitoring transmembrane potential. The microfluidic device includes a body structure having at least one microscale cavity (e.g., microchannel, microchamber, or the like) disposed therein. A target source of a first composition which includes at least one membrane is fluidly coupled to the at least one microscale cavity (e.g., a microscale channel, chamber, well, column or the like). A cationic membrane permeable staining dye source which includes one or more cationic membrane permeable nucleic acid staining dye, is fluidly coupled to the at least one microscale cavity. Alternatively or in addition, an anionic membrane permeable redistributing dye source which includes one or more anionic redistributing dye is fluidly coupled to the at least one microscale cavity. During operation of the device, the first composition is contacted, in the presence of the cationic membrane permeable staining dye, and/or the anionic membrane permeable redistributing dye, to at least one transmembrane potential modulatory composition.
In applications where the device is used for screening effects of modulatory compositions, the device can include a source of at least one potential membrane modulatory composition fluidly coupled to the at least one microscale cavity. The potential membrane modulatory composition can be, e.g., a membrane hyperpolarization buffer, a membrane depolarization buffer, or a compound which alters ionic permeability of the membrane.
The device typically includes a signal detector located proximal to the microscale cavity. The signal detector detects the detectable signal, e.g., for a selected length of time (t). For example, the detector can include a spectrophotometer, or an optical detection element. Commonly, the signal detector is operably coupled to a computer, which deconvolves the detectable signal to provide an indication of the transmembrane potential, e.g., an indication of a change in the potential over time.
In one typical embodiment, during operation of the device, the first composition comprising at least one membrane is flowed from the target source into the cavity, e.g., into a microchannel. The potential membrane modulatory composition is flowed from the target source into contact with the first composition. The cationic membrane permeable staining dye and/or an anionic membrane permeable redistributing dye is flowed into contact with the first composition and the detectable signal is monitored at a selected time (t) after contact of the first composition with the cationic membrane permeable staining dye and/or the an anionic membrane permeable redistributing dye.
In one aspect, the invention provides a composition, e.g., for practicing the methods noted above. The composition includes a first component comprising a membrane, a cationic membrane permeable nucleic acid staining dye, and an anionic membrane permeable redistributing dye. The membrane component can include, e.g., a cell, mitochondria, chloroplast, cell vesicle, a membrane preparation of a cell or cell component, or an artificial membrane. The cell can be an intact cell which can be, e.g., an animal cell, a plant cell, a fungal cell or a bacterial cell. For example, the cell can be any of those noted herein.
Similarly, the cationic membrane permeable nucleic acid staining dye and the anionic membrane permeable redistributing dye can be any of those noted above with reference to the methods of the invention. The composition can also include buffers, ions, etc., as noted herein. A container or microfluidic processor comprising the composition is also a feature of the invention. For example, the composition of the invention can be present in a kit or microfluidic processor. The kit can additionally include, e.g., instructions for practicing the method of the invention, control compounds, test compounds, containers for holding reagents, packaging materials, or the like.